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John R Hassell - One of the best experts on this subject based on the ideXlab platform.
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IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro.
Experimental Eye Research, 2007Co-Authors: Kurt Musselmann, Bradley Kane, Bridgette Alexandrou, John R HassellAbstract:Abstract Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-β/ml over a 7-day culture period. We found that IGF-II and TGF-β, alone or combined, increased both 3 H-thymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to α-SM (smooth muscle) actin, fibronectin, SPARC, lumican and Keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no α-SM actin or fibronectin, low levels of SPARC and high levels of lumican and Keratocan, indicating a native phenotype. Keratocytes in TGF-β expressed α-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of Keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-β, however, expressed α-SM actin, fibronectin, SPARC, lumican, and Keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-β mediated down regulation of Keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-β.
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Stimulation of collagen synthesis by insulin and proteoglycan accumulation by ascorbate in bovine keratocytes in vitro.
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Kurt Musselmann, Bradley Kane, Bridgette Alexandrou, John R HassellAbstract:PURPOSE. Ascorbate is required for the hydroxylation of collagen that is present in the corneal stroma. The keratan sulfate proteoglycans (KSPGs) lumican and Keratocan are also present, and they interact with collagen and modulate its assembly into fibrils. In this study, ascorbate was added to a defined medium containing insulin, and its effects on the synthesis of collagen and KSPGs by keratocytes were determined. METHODS. Collagenase-isolated keratocytes were cultured with or without insulin with or without ascorbate. Collagen and glycosaminoglycan synthesis was determined by collagenase digestion of incorporated 3 H-glycine and by chondroitinase ABC or endo-β-galactosidase digestion of incorporated 35 SO 4 . KSPGs were detected by Western blot. Collagen stability was determined by pepsin digestion. Ethyl-3,4-dihydroxybenzoate (EDB) was used to inhibit collagen hydroxylation. RESULTS. Insulin stimulated the synthesis of collagen but did not affect the accumulation of lumican and Keratocan. Insulin plus ascorbate, however, stimulated the synthesis of collagen and increased the accumulation of these proteoglycans. The accumulation of PGDS, a KSPG that does not interact with collagen, was not affected by ascorbate. Only the collagen synthesized in the presence of ascorbate was pepsin resistant. EDB overrode the effects of ascorbate on pepsin resistance and proteoglycan accumulation. CONCLUSIONS. The results of this study indicate that the accumulation of lumican and Keratocan depends in part on the level of collagen synthesis and its hydroxylation. The interaction of lumican and Keratocan with the stably folded triple helix provided by hydroxylation may also serve to stabilize these proteoglycans.
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Maintenance of the keratocyte phenotype during cell proliferation stimulated by insulin.
Journal of Biological Chemistry, 2005Co-Authors: Kurt Musselmann, Bradley Kane, Bridgette Alexandrou, John R HassellAbstract:Abstract Keratocytes normally express high levels of aldehyde dehydrogenase and Keratocan. They proliferate and lose their keratocyte markers when they become fibroblastic during corneal wound healing. Keratocytes cultured in fetal bovine serum also become fibroblastic, proliferate, and lose these markers. In this report, we studied the effects of three serum growth factors, fibroblast growth factor-2, insulin, and platelet-derived growth factor-BB, on keratocyte proliferation and the maintenance of the keratocyte markers in 7-day cultures in cells plated at low (5,000 cells/cm2) and high (20,000 cells/cm2) density in serum-free medium. Keratocyte proliferation was measured by [3H]thymidine incorporation and by DNA content of the cultures. Cytosolic aldehyde dehydrogenase and Keratocan accumulated in the medium were quantified by Western blot. The results showed that all the growth factors stimulated proliferation, but insulin stimulated proliferation more consistently. The keratocyte markers aldehyde dehydrogenase and Keratocan were maintained after 7 days in culture in all growth factors, but keratocyte cell morphology was only maintained in medium containing insulin. Most of the proteoglycans were degraded in cultures of keratocytes plated at low density and cultured in the absence of growth factors. This degradation was prevented when keratocytes were cultured in the presence of the growth factors or when keratocytes were plated at high density. The results of this study show that insulin can expand keratocytes in vitro, maintain their phenotype, and prevent proteoglycan degradation.
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Keratocan deficient mice display alterations in corneal structure
Journal of Biological Chemistry, 2003Co-Authors: David E Birk, John R Hassell, Bradley KaneAbstract:Abstract Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or Keratocan protein was detected in the Kera–/– cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera–/– cornea as compared with that of the wild-type littermates. Mice lacking Keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera–/– mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.
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Production of prostaglandin D synthase as a keratan sulfate proteoglycan by cultured bovine keratocytes.
Investigative Ophthalmology & Visual Science, 2001Co-Authors: Bridgette L Berryhill, Marianne P Beales, John R HassellAbstract:PURPOSE: To characterize the major proteoglycans produced and secreted by collagenase-isolated bovine keratocytes in culture. METHODS: Freshly isolated keratocytes from mature bovine corneas were cultured in serum-free Dulbecco's modified Eagle's medium/ F12. Secreted proteoglycans were radiolabeled with protein labeling mix ((35)S-Express; Dupont NEN Life Science Products, Boston, MA) and digested with chondroitinase ABC, keratanase, and endo-beta-galactosidase to remove glycosaminoglycan chains, and core proteins were analyzed by autoradiography and Western blot analysis. An unidentified keratan sulfate proteoglycan (KSPG) was purified by gel filtration (Superose 6; Amersham Pharmacia, Piscataway, NJ) and anion-exchange chromatography (Resource Q; Amersham Pharmacia) and subjected to amino acid sequencing. RESULTS: Keratanase digestion of proteoglycans produced approximately 50 kDa core proteins that immunoreacted with antisera to lumican, Keratocan, and osteoglycin-mimecan. Chondroitinase ABC digestion produced a approximately 55-kDa core protein that immunoreacted with antisera to decorin. A 28-kDa band generated by keratanase or endo-beta-galactosidase digestion did not react with these antibodies. Chromatographic purification and amino acid sequencing revealed that the protein was prostaglandin D synthase (PGDS). Identity was confirmed by Western blot analysis using antisera to recombinant PGDS. PGDS isolated from corneal extracts was not keratanase sensitive but was susceptible to endo-beta-galactosidase, suggesting that it contains unsulfated polylactosamine chains in native tissue and is therefore present as a glycoprotein. CONCLUSIONS: These results indicate that bovine keratocytes, when cultured under serum-free conditions, produce the four known leucine-rich proteoglycans decorin, Keratocan, lumican, and osteoglycin/mimecan and maintain a phenotype that is comparable to that of in situ keratocytes. Additionally, these cells produce PGDS, a known retinoid transporter, as a KSPG.
Ijong Wang - One of the best experts on this subject based on the ideXlab platform.
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Morphological Differences between the Trabecular Meshworks of Zebrafish and Mammals
Current Eye Research, 2020Co-Authors: Chun-chen Chen, Fungrong Hu, John R. Samples, Ijong WangAbstract:Purpose: The zebrafish has been used as an animal model to study ocular development and diseases, including glaucoma. However, there are still many concerns about the morphological differences between zebrafish and mammals. Before using the zebrafish for glaucoma studies, we should understand the morphological differences in the trabecular meshworks (TMs) of zebrafish and other animal models. This study investigated and compared the histological morphologies and compositions of the extracellular matrices of the TMs of the zebrafish and some commonly used animal models, including the mouse, rat, rabbit, and cow. Methods: Sections of the angular portions from the studied species (mouse, rat, rabbit, cow, zebrafish, and human) were prepared for immunohistochemical and electron microscopic analyses. Antibodies directed against cytoskeletal and extracellular matrix components (AE1/AE3, vimentin, α-smooth muscle actin, Keratocan, and lumican) were used for immunolocalization. Reverse transcription polymerase ch...
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Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice.
PLOS ONE, 2010Co-Authors: Jianhua Zhang, Ijong Wang, Martin Sieber, John T. Chang, James V JesterAbstract:BackgroundKeratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation.Methodology/Principal FindingsHuman mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated Keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of Keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice.Conclusions/SignificanceUmbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.
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Cell Therapy of Congenital Corneal Diseases with Umbilical Mesenchymal Stem Cells: Lumican Null Mice - eScholarship
2010Co-Authors: Jianhua Zhang, Ijong Wang, Martin Sieber, John T. Chang, James V JesterAbstract:BackgroundKeratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation.Methodology/Principal FindingsHuman mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated Keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of Keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice.Conclusions/SignificanceUmbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.
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molecular analysis and characterization of zebrafish Keratocan zkera gene
Journal of Biological Chemistry, 2008Co-Authors: Chungliang Chien, Richard Converse, Muhshy Chen, Fungrong Hu, Fonjou Hsieh, Ijong WangAbstract:Abstract Corneal small leucine-rich proteoglycans play a pivotal role in maintaining corneal transparency and function. In this study, we isolated and characterized the zebrafish (Danio rerio) Keratocan (zKera) gene. The human Keratocan sequence was used to search zebrafish homologues. The zKera full-length genomic DNA and cDNA were generated via PCR of zebrafish genomic DNA and reverse transcription-PCR of total zebrafish eye RNA, respectively. The zKera spanning 3.5 kilobase pairs consists of two exons and one intron and a TATA-less promoter. The zKera encodes 341 amino acids with 59% identity to its human counterpart and 57% identity to that of mouse Keratocan. Like mouse and chick Keratocan, zKera mRNA is selectively expressed in the adult cornea; however, during embryonic development, zKera mRNA is expressed in both the brain and the cornea. Interestingly, it is expressed mainly in corneal epithelium but also in the stroma. A pseudogene was proved by introducing a zKera promoter-driven enhanced green fluorescence protein reporter gene into fertilized zebrafish eggs. Using morpholino-antisense against zKera to knock down zKera resulted in a lethal phenotype due to massive caspase-dependent apoptosis, which was noted by a significant increase of active caspase-3 and caspase-8 in the developing forebrain area, including the eyes. This is different from mouse, for which Keratocan-deficient mice are viable. Taken together, our data indicate that mammalian Keratocan is conserved in zebrafish in terms of gene structure, expression pattern, and promoter function.
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Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-α signaling
Developmental Biology, 2005Co-Authors: Yasuhito Hayashi, James L Funderburgh, Ijong Wang, Shizuya Saika, James J. Jester, M. Hayashi, Peter J. RoughleyAbstract:Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix (ECM) in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development. In this study, we showed that transgenic mice overexpressing biglycan under the Keratocan promoter exhibited exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation of eyelid muscle formation and the failure of meibomian gland formation. In addition, in vitro analysis revealed that biglycan binds to TGF-α, thus interrupting EGFR signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGF-α signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of HB-EGF expression elicited by TGF-α. These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGF-α gradient serving as a morphogen of eyelid morphogenesis.
Gary W Conrad - One of the best experts on this subject based on the ideXlab platform.
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Effects of Ultraviolet-A and Riboflavin on the Interaction of Collagen and Proteoglycans during Corneal Cross-linking
Journal of Biological Chemistry, 2011Co-Authors: Yuntao Zhang, Abigail H Conrad, Gary W ConradAbstract:Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains Keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both Keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both Keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and Keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that Keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.
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An x-ray diffraction study of corneal structure in mimecan-deficient mice.
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Nicola Beecher, Gary W Conrad, Keith Michael Andrew Meek, Connie Carlson, Bryan R. Allen, Rogers Kipchumba, Andrew J. QuantockAbstract:PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and Keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and Keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea.
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the Keratocan gene is expressed in both ocular and non ocular tissues during early chick development
Matrix Biology, 2003Co-Authors: Abigail H Conrad, Gary W ConradAbstract:Abstract Extracellular matrix (ECM) keratan sulfate proteoglycans (KSPGs) are core proteins with sulfated polylactosamine side chains (KS). The KSPG core protein Keratocan gene ( Kera ) is expressed almost exclusively in adult vertebrate cornea, but its embryonic expression is little known. Embryonic chick in situ hybridization reveals Kera mRNA expression in corneal endothelium from embryonic day (E) 4.5, Hamburger–Hamilton (HH) 25, in stromal keratocytes from E6.5, HH30, and in iris distal surface cells from E8, HH34. As highly sulfated, antibody I22-positive KS increases extracellularly from posterior to anterior across the stroma, nerves enter and populate only anterior stroma and epithelium. RT-PCR and in situ hybridization demonstrate that developmentally regulated Kera mRNA expression initiates in midbrain and dorsolateral mesenchyme at E1, HH7, then spreads caudally in hindbrain and cranial and trunk mesenchyme flanking the neural tube through E2, HH20. Cranial expression extends ventrally through the developing head, and concentrates in mesenchyme surrounding eye anterior regions and cranial ganglia, and in subepidermal pharyngeal arch mesenchyme by E3.5, HH22. Kera expression in the trunk at E3.5, HH22 and E4.5, HH25, is strong in dorsolateral subepidermal, sclerotomal and nephrogenic mesenchymes, but absent in neural tube, dorsal root ganglia, nerve outgrowths, notochord, heart and gut. Early limb buds express Kera mRNA throughout their mesenchyme, then in restricted proximal and distal mesenchymes. I22-positive KS appears only in notochord in E3.5, HH22 and E4.5, HH25, embryos. Results suggest the hypothesis that Keratocan, or Keratocan with minimally sulfated KS chains, may play a role in structuring ECM for early embryonic cell and neuronal migrations.
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molecular cloning and relative tissue expression of Keratocan and mimecan in embryonic quail cornea
Matrix Biology, 2000Co-Authors: Lolita M Corpuz, Jane R Dunlevy, John R Hassell, Abigail H Conrad, Gary W ConradAbstract:We have cloned and sequenced the cDNAs for quail cornea keratan sulfate proteoglycan core proteins, Keratocan and mimecan. The deduced quail Keratocan protein contains a single conservative amino acid difference from the chick sequence, whereas quail mimecan protein contains a 58 amino acid-long avian-unique sequence that shares no homology with mammalian mimecan. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that Keratocan and lumican are expressed at highest levels in cornea, whereas mimecan mRNA is expressed at a much lower level. Keratocan is expressed only in quail cornea, whereas mimecan is expressed in many different tissues as four transcripts of different sizes. Both lumican and mimecan are expressed at lowest levels in brain, liver and sternum.
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identification and characterization of conserved cis regulatory elements in the human Keratocan gene promoter
Biochimica et Biophysica Acta, 2000Co-Authors: Elena S Tasheva, Abigail H Conrad, Gary W ConradAbstract:Abstract Keratocan, along with lumican and mimecan, represent the keratan sulfate-containing proteoglycans of the vertebrate cornea that play a key role in development and maintenance of corneal transparency. In this study, we cloned 4.1 kb of the human Kera 5′-flanking region and characterized the promoter structure. Using primer extension and ribonuclease protection assay, we identify two major transcriptional start sites in the first exon. Using luciferase reporter gene transfection analysis of 5′-deletion and mutation constructs, we demonstrate positive and negative regulatory elements within a 1.3 kb upstream sequence. Comparison of human and bovine 5′-flanking sequences reveals three highly conserved regions: a 450 bp region in the first exon, a 92 bp promoter proximal conserved regulatory region identified as an enhancer in the natural context, and a 223 bp promoter distal conserved regulatory region identified as a silencer both in the natural context and in a heterologous promoter system. In addition, a conserved CArG-box residing 851 bp upstream of the first transcription start site also can lead to the repression of Kera expression in cultured corneal keratocytes. DNaseI footprinting and electrophoretic mobility shift assay demonstrate that cell type-specific factors bind to regulatory elements located in the conserved regions. Competition experiments indicate that the CTC factor and a protein that binds to the CAGA motif are likely to be among the multiple factors involved in the transcriptional regulation of the human Kera gene.
Scheffer C G Tseng - One of the best experts on this subject based on the ideXlab platform.
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Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-β Signaling in a Low-Calcium, Serum-Free Medium
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Tetsuya Kawakita, Jean-marie A Parel, Edgar M Espana, Hua He, Robert Smiddy, Scheffer C G TsengAbstract:Keratocytes, a unique population of neural crest-derived cells embedded in the corneal stroma, play a major role in maintaining corneal transparency. They are mitotically quiescent, exhibit a dendritic morphology with extensive intercellular contacts,1,2 and express keratan sulfate-containing proteoglycans (KSPGs),3,4 aldehyde dehydrogenase (ALDH)4 and CD34.5–7 When a scar forms during corneal wound healing, keratocytes turn into fibroblasts by losing the aforementioned morphology and downregulating the expression of KSPG8,9 and CD34,5 and eventually they differentiate into myofibroblasts10,11 that express α-smooth muscle actin (α-SMA), fibronectin, and biglycan.4 The aforementioned abnormal phenotypic changes can be observed in vitro on plastic dishes by adding fetal bovine serum (FBS). For example, the characteristic dendritic morphology of bovine,12 rabbit,13,14 and human7,15 keratocytes is maintained in a serum-free medium. Nevertheless, when FBS is added, cells take on a flattened fibroblastic morphology and lose the expression of KSPG, including corneal stroma-specific Keratocan.4,7,15–19 Unfortunately, keratocytes cultured in the serum-free medium do not proliferate. Therefore, expanding keratocytes while maintaining their normal phenotype in vitro is very desirable. Because FBS contains TGF-β,20 which promotes myofibroblast differentiation,4,14,21,22 many have speculated that TGF-β signaling is responsible for promoting myofibroblast differentiation from keratocytes. It remains unknown whether suppression of TGF-β signaling is not only sufficient to prevent myofibroblast differentiation but also essential for maintaining the keratocyte phenotype. Because expression of the TGF-β2, -β3, and -β RII transcripts is suppressed when human corneal and limbal fibroblasts23 and human conjunctival and pterygium fibroblasts24 were cultured on human amniotic membrane (AM) stroma, we used AM as a culturing substrate to maintain a dendritic morphology and expression of Keratocan and CD34 by human keratocytes while continuously stimulating them to proliferate in a medium containing 10% FBS.7,15 Extracellular calcium concentration ([Ca2+]) significantly affects TGF-β signaling. For example, increased [Ca2+] promotes expression of TGF-β in human vascular endothelial cells.25 Chelation of extracellular [Ca2+] blocks TGF-β-mediated [Ca2+] influx and calcineurin (calcium-dependent signaling intermediate) activity,26 and inhibits TGF-β-mediated α-SMA promoter signaling in human embryonic lung fibroblasts.27 With this [Ca2+] effect in mind, we examined in the current study whether the phenotype of keratocytes can also be maintained on plastic by suppressing TGF-β signaling with a low-[Ca2+], serum-free medium supplemented with growth factors.
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The Heterogeneous Murine Corneal Stromal Cell Populations In Vitro
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Edgar M Espana, Jean-marie A Parel, Tetsuya Kawakita, Mario A Di Pascuale, Wei Li, Scheffer C G TsengAbstract:PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), Keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed Keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.
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Keratocan expression of murine keratocytes is maintained on amniotic membrane by down regulating transforming growth factor β signaling
Journal of Biological Chemistry, 2005Co-Authors: Tetsuya Kawakita, Edgar M Espana, Hua He, Armand Hornia, J Ouyang, Scheffer C G TsengAbstract:Keratocytes, a unique population of neural crest-derived cells embedded in the corneal stroma, play a major role in maintaining corneal transparency. Different culturing methods have been explored to study the mechanism whereby normal keratocytes are regulated in vitro. Bovine (1), rabbit (2) and human (3;4) keratocytes cultured on plastic rapidly lose their characteristic dendritic morphology, and acquire a fibroblastic morphology when exposed to a fetal bovine serum (FBS)-containing medium. Furthermore, these exposed cells downregulate the expression of keratan sulfate-containing proteoglycans (5–8), Keratocan (3;9), and CD34 (4), and upregulate that of chondroitin-dermatan sulfate-containing proteoglycans (9;10) and α-SMA (2;4;9;11). Similarly, murine corneal fibroblasts cultured on plastic in a FBS-containing medium lose expression of corneal stroma-specific Keratocan mRNA, but continue to express mimecan and lumican mRNAs (12;13). To overcome such a detrimental effect of FBS, a serum-free culture system has to be used to maintain a normal dendritic morphology and expression of keratan sulfate-containing proteoglycans at the expense of cell expansion (1). TGF-β signaling is essential for corneal development and plays a pivotal role in normal and abnormal wound healing (14;15). One important mediator of TGF-β signaling is the Smad pathway. The binding of TGF-β• • ligands • • to TGF-β • • receptors triggers phosphorylation of Smad 2 and Smad 3. Smad 4 binds with phosphorylated Smad 3 in a complex and is translocated to the nucleus to transactivate target genes (16). Therefore, the translocation of these Smads into the nucleus is a common effector in the Smad-mediated pathway (17). In 1% serum or a serum-free medium, addition of TGF-β1 differentiates keratocytes into myofibroblasts with prominent focal adhesions, and upregulates expression of α-SMA, integrin α5β1, cadherins, type I and III collagens, biglycan and the EDA (EIIIA) form of fibronectin (9–11;18). It remains unclear whether suppression of TGF-β signaling is not only important to prevent myofibroblast differentiation but also essential for maintaining the normal keratocyte phenotype. Recently, we reported a culture system that can achieve effective expansion of human keratocytes by growing them on the stromal surface of the AM in an FBS-containing medium (3;4). They maintained a dendritic morphology, continued to express corneal stroma-specific Keratocan for at least 5 passages (at 1:2 split), and did not express α-SMA under serum-containing conditions or addition of TGF-β1 (4). Previously, we have reported that transcript expression of TGF-β2, TGF-β3, and TGF-βRII is suppressed in both serum-containing and serum-free media, of which the latter is challenged by exogenous TGF-β1, in cultured human corneal and limbal fibroblasts (19) as well as human conjunctival and pterygium fibroblasts (20). Suppression of TGF-β signaling is coupled with downregulation of α-SMA, integrin α5 and EDA form of fibronectin transcripts. Herein, we report that murine keratocytes can be similarly expanded on AM for at least 8 passages without losing their normal phenotype in a serum-containing medium and that suppression of Smad-mediated TGF-β signaling pathway is pivotal in maintaining Keratocan-expressing phenotype.
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human keratocytes cultured on amniotic membrane stroma preserve morphology and express Keratocan
Investigative Ophthalmology & Visual Science, 2003Co-Authors: Edgar M Espana, Hua He, Tetsuya Kawakita, Mario A Di Pascuale, V K Raju, Scheffer C G TsengAbstract:PURPOSE: To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and Keratocan expression. METHODS: Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of Keratocan was determined by RT-PCR and Western blot analysis. RESULTS: Trephined stroma yielded 91,600 +/- 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of Keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize Keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS: Human keratocytes maintain their characteristic morphology and Keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.
James L Funderburgh - One of the best experts on this subject based on the ideXlab platform.
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3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture
PLOS ONE, 2017Co-Authors: Chiara E. Ghezzi, James L Funderburgh, Benedetto Marelli, Fiorenzo G. Omenetto, David L. KaplanAbstract:The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and Keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including Keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single silk films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.
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Dental Pulp Stem Cells: A New Cellular Resource for Corneal Stromal Regeneration
Stem Cells Translational Medicine, 2015Co-Authors: Fatima N. Syed-picard, Martha L Funderburgh, Yiqin Du, Kira L. Lathrop, Mary M. Mann, James L FunderburghAbstract:Corneal blindness afflicts millions of individuals worldwide and is currently treated by grafting with cadaveric tissues; however, there are worldwide donor tissue shortages, and many allogeneic grafts are eventually rejected. Autologous stem cells present a prospect for personalized regenerative medicine and an alternative to cadaveric tissue grafts. Dental pulp contains a population of adult stem cells and, similar to corneal stroma, develops embryonically from the cranial neural crest. We report that adult dental pulp cells (DPCs) isolated from third molars have the capability to differentiate into keratocytes, cells of the corneal stoma. After inducing differentiation in vitro, DPCs expressed molecules characteristic of keratocytes, Keratocan, and keratan sulfate proteoglycans at both the gene and the protein levels. DPCs cultured on aligned nanofiber substrates generated tissue-engineered, corneal stromal-like constructs, recapitulating the tightly packed, aligned, parallel fibrillar collagen of native stromal tissue. After injection in vivo into mouse corneal stroma, human DPCs produced corneal stromal extracellular matrix containing human type I collagen and Keratocan and did not affect corneal transparency or induce immunological rejection. These findings demonstrate a potential for the clinical application of DPCs in cellular or tissue engineering therapies for corneal stromal blindness.
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Differentiation of human embryonic stem cells into cells with corneal keratocyte phenotype.
PLOS ONE, 2013Co-Authors: Audrey A. Chan, Martha L Funderburgh, Yiqin Du, Mary M. Mann, Andrew J. Hertsenberg, Katherine Davoli, Jocelyn D. Mich-basso, Lei Yang, James L FunderburghAbstract:Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for Keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight Keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.
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Keratocyte phenotype is enhanced in the absence of attachment to the substratum.
Molecular Vision, 2008Co-Authors: Martha L Funderburgh, Mary M. Mann, James L FunderburghAbstract:PURPOSE: Keratocytes, mesenchymal cells populating the corneal stroma, secrete the unique transparent connective tissue of the cornea as well as opaque scar tissue after injury. Previous studies identified factors mediating keratocyte phenotype in vitro, particularly the expression of the keratan sulfate proteoglycans, which are essential for vision. Whereas earlier work emphasized effects of cytokines, the current study examines the effects of substratum attachment on keratocyte phenotype. METHODS: Primary keratocytes from collagenase digestion of bovine corneas were cultured on tissue-culture plastic or on poly (2-hydroxyethylmethacrylate)(polyHEMA)-coated, non-adhesive surfaces. Secreted proteoglycans from culture media and cell-associated proteins were characterized using western blotting or isotopic labeling. Gene expression was characterized with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Secreted matrix was examined with immunostaining. RESULTS: We observed that virtually all primary keratocytes participate in the formation of spheroidal aggregates, remaining viable for at least four weeks in vitro. Spheroid keratocytes secrete more keratan sulfate and Keratocan than attached cells in the same culture medium. In spheroids, keratocytes accumulate substantial matrix in intercellular spaces, including keratan sulfate, lumican, Keratocan, and collagens V and VI. The unattached cells undergo limited cell division and do not differentiate into myofibroblasts in response to transforming growth factor beta (TGFbeta), which is based on the expression of extra domain A (EDA) fibronectin and alpha-smooth muscle actin. Similarly, the platelet derived growth factor, a cytokine initiating the fibroblastic phenotype in attached keratocytes, had a limited effect on the spheroid-associated keratocytes. Ascorbate-2-phosphate was the only agent stimulating keratan sulfate secretion in the spheroid keratocytes. CONCLUSIONS: These results provide a new paradigm for understanding signals that regulate extracellular matrix secretion. For primary keratocytes, the alteration of the cellular environment in terms of cell-cell and cell-matrix interactions mediates and can override signals from soluble cytokines in influencing matrix expression and also in adopting other aspects of the fibroblastic and myofibroblastic phenotypes found in healing wounds.
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Keratocan a cornea specific keratan sulfate proteoglycan is regulated by lumican
Journal of Biological Chemistry, 2005Co-Authors: Eric C Carlson, James L Funderburgh, Taiichiro Chikama, Yasuhito Hayashi, David E Birk, James V JesterAbstract:Abstract Lumican is an extracellular matrix glycoprotein widely distributed in mammalian connective tissues. Corneal lumican modified with keratan sulfate constitutes one of the major proteoglycans of the stroma. Lumican-null mice exhibit altered collagen fibril organization and loss of corneal transparency. A closely related protein, Keratocan, carries the remaining keratan sulfate of the cornea, but Keratocan-null mice exhibit a less severe corneal phenotype. In the current study, we examined the effect of lumican overexpression in corneas of wild type mice. These mice showed no alteration in collagen organization or transparency but had increased Keratocan expression at both protein and mRNA levels. Corneas of lumican-null mice showed decreased Keratocan. This coupling of Keratocan expression with lumican also was observed after intrastromal injection of a lumican expression minigene into the corneal stroma of Lum–/– mice. Small interfering RNA knockdown of lumican in vitro reduced Keratocan expression, whereas co-injection of a lumican-expressing minigene with a β-galactosidase reporter driven by the Keratocan promoter demonstrated an increase of Keratocan transcriptional activity in response to lumican expression in Lum–/– corneas in vivo. These observations demonstrate that lumican has a novel regulatory role in Keratocan expression at the transcriptional level. Such results help provide an explanation for the differences in severity of corneal manifestation found in Lum–/– and Kera–/– mice. The results also suggest a critical level of small proteoglycans to be essential for collagen organization but that overabundance is not detrimental to extracellular matrix morphogenesis.
